The present invention is generally in the area of regeneration and repair of cartilage, and more particularly relates to implantation of mesenchymal stem cells on matrices to form cartilage.
Arthritis, both rheumatoid and osteoarthritis, constitutes a major medical problem. In particular, degeneration of articular cartilage in osteoarthritis is a serious medical problem. Drugs are given to control the pain and to keep the swelling down, but the cartilage continues to be destroyed. Eventually, the joint must be replaced. As reviewed by Mankin, N. E. J. Med. 331(14), 940-941 (October 1994), it is still unknown why cartilage does not heal and no solutions to this problem are known.
Whether articular cartilage is damaged from trauma or congenital anomalies, its successful clinical regeneration is poor at best, as reviewed by Howell, et al. Osteoarthritis: Diagnosis and Management, 2nd ed., (Philadelphia, W. B. Saunders, 1990) and Kelley, et al. Textbook of Rheumatology, 3rd ed., (Philadelphia, W. B. Saunders, 1989) 1480. The inability of adult articular cartilage for self repair has been well recognized and has stimulated major interest. There are two major mechanisms of articular cartilage repair: intrinsic and extrinsic, discussed by Edwards Proc. Ins. Mech. Eng. 181, 16 (1967), and Sokoloff J. Rheumatol. 1, 1 (1974).
Superficial or partial-thickness injuries that do not penetrate the subchondral bone rely on the intrinsic mechanism for repair. Soon after superficial injury, chondrocytes adjacent to the injured surfaces show a brief burst of mitotic activity associated with an increase in glycosaminoglycan and collagen synthesis. Despite these attempts at repair, there is no appreciable increase in the bulk of cartilage matrix and the repair process is rarely effective in healing the defects.
Osteochondral, or full-thickness, cartilage defects extend into the subchondral bone. Such defects arise after the detachment of osteochondritic dissecting flaps, fractured osteochondral fragments, or from chronic wear of degenerative articular cartilage. Osteochondral defects depend on the extrinsic mechanism for repair. Extrinsic healing relies on mesenchymal elements from subchondral bone to participate in the formation of new connective tissue. This fibrous tissue may or may not undergo metaplastic changes to form fibrocartilage. Even if fibrocartilage is formed, it does not display the same biochemical composition or mechanical properties of normal articular cartilage or subchondral bone and degenerates with use, Furukawa, et al., J. Bone Joint Surg. 62A, 79 (1980); Coletti, et al., J. Bone Joint Surg. 54A, 147 (1972); Buckwalter, et al., "Articular cartilage: composition, structure, response to injury and methods of facilitating repair", in Articular Cartilage and Knee Joint Function: Basic Science and Arthroscopy, Ewing J E, Ed., (New York, Raven Press, 1990), 19. The ensuing osteoarthritis may result in permanent disability and discomfort to the patient.
As described in U.S. Pat. No. 5,041,138 to Vacanti, et al., and U.S. Pat. No. 4,846,835 to Grande, cartilage has been grown by seeding synthetic polymeric matrices with dissociated cells, which are then implanted to form new cartilage. Cartilage has also been grown from an injected or implanted ionically crosslinked hydrogel-chondrocyte suspension, as described by Atala, et al., J. Urology vol. 150, no. 2, part 2, 745-747 (August 1993). Injection of dissociated chondrocytes directly into a defect has also recently been described as a means for forming new cartilage, as reported by Brittberg, et al., N. E. J. Med. 331,889-895 (October 1994). Cartilage was harvested from minor load-bearing regions on the upper medial femoral condyle of the damaged knee, cultured, and implanted two to three weeks after harvesting.
Freed and Grande, J. Biomed. Mater. Res. 28, 891 (1994) cultured mature chondrocytes from New Zealand white rabbits in vitro onto polyglycolic acid (PGA) scaffolds for 21/2 weeks. A full thickness articular cartilage defect was then created in the femoropatellar groove bilaterally in syngeneic New Zealand white rabbits. Mature chondrocytes on the PGA-matrix (PGA-cells) were imbedded into one knee joint while PGA discs alone were imbedded into the contralateral knee and the animals euthanized at one and six months post-implantation. The repair tissue was well bonded to the host tissue and the surfaces of these defects were congruent with the host cartilage. The PGA alone showed a mixture of fibrocartilage and hyaline cartilage oriented randomly. The PGA-cells implant showed normal articular cartilage histology, but did not have normal subchondral bone.
A disadvantage of these systems is that the chondrocytes must be obtained from the patient, typically by a biopsy, cultured, and then implanted on the matrix. This is relatively easy in laboratory animals, but presents greater logistical problems in humans where a defect is created by the biopsy required to provide cells for repair of another defect. Moreover, if the defect includes a part of the underlying bone, this is not corrected using chondrocytes, which are already differentiated and will not form new bone. The bone is required to support the new cartilage.
Stem cells are defined as cells which are not terminally differentiated, which can divide without limit, and divides to yield cells that are either stem cells or which irreversibly differentiate to yield a new type of cell. Those stem cells which give rise to a single type of cell are call unipotent cells; those which give rise to many cell types are called pluripotent cells. Chondro/osteoprogenitor cells, which are bipotent with the ability to differentiate into cartilage or bone, were isolated from bone marrow (for example, as described by Owen, J. Cell Sci. Suppl. 10, 63-76 (1988) and in U.S. Pat. No. 5,226,914 to Caplan, et al.). These cells led Owen to postulate the existence of pluripotent mesenchymal stem cells, which were subsequently isolated from muscle (Pate, et al., Proc. 49th Ann. Sess. Forum Fundamental Surg. Problems 587-589 (Oct. 10-15, 1993)), heart (Dalton, et al., J. Cell Biol. 119, R202 (March 1993)), and granulation tissue (Lucas, et al., J. Cell Biochem. 122, R212 (March 1993)). Pluripotency is demonstrated using a non-specific inducer, dexamethasone (DMSO), which elicits differentiation of the stem cells into chondrocytes (cartilage), osteoblasts (bone), myotubes (muscle), adipocytes (fat), and connective tissue cells.
Unfortunately, although it is highly desirable to have stem cells which are easily obtained by a muscle biopsy, cultured to yield large numbers, and can be used as a source of chondrocytes or osteoblasts or myocytes, there is no known specific inducer of the mesenchymal stem cells that yields only cartilage. In vitro studies in which differentiation is achieved yields a mixture of cell types. Studies described in U.S. Pat. Nos. 5,226,914 and 5,197,985 to Caplan, et al., in which the cells were absorbed into porous ceramic blocks and implanted yielded primarily bone. Studies using bone morphogenic protein-2 (rhBMP-2) in vivo always yield an endochondral bone cascade. That is, cartilage is formed first, but this cartilage hypertrophies, is invaded by vasculature and osteoblasts, and is eventually replaced by bone complete with marrow (Wozney, Progress in Growth Factor Research 1, 267-280 (1989)). Studies testing rhBMP-2 on the mesenchymal stem cells in vitro produced mixtures of differentiated cells, although cartilage predominated (Dalton, et al., J. Cell Biol. 278, PZ202 (February 1994)). Incubation of mesenchymal cell cultures with insulin led to a mixed myogenic and adipogenic response, while incubation with insulin-like growth factors I or II led to a primarily myogenic response (Young, et al., J. Cell Biochem. 136, CD307 (April 1992)). U.S. Pat. Nos. 4,774,322 and 4,434,094 to Seyedin, et al., report the isolation of a factor that induces an osteogenic response in vivo or cartilage formation in vitro when mixed with muscle cells.
It is therefore an object of the present invention to provide a method and compositions for formation of cartilage.
It is a further object of the present invention to provide a method and compositions to induce differentiation of mesenchymal stem cells into cartilage.